Biosensors utilizing ligand induced conformation changes

ABSTRACT

A method for detecting the presence of a target analyte comprising binding a target analyte to a binding ligand comprising at least a first electron donor moiety and a second electron acceptor moiety; and detecting the electron transfer between the donor and acceptor, wherein there is a change in the amount of electron transfer between the donor and acceptor as a result of altering the structured state of the donor and acceptor caused by a conformational change in the binding ligand upon binding of the target ligand.

This is a continuation of application Serial No. 60/116,893 filed onJan. 22, 1999.

FIELD OF THE INVENTION

The invention relates to the detection of ligands that causeconformational changes in their binding partners upon binding. As aresult of the conformational change, electron transfer between twoelectron transfer moieties can occur.

BACKGROUND OF THE INVENTION

There are a number of assays and sensors for the detection of thepresence and/or concentration of specific substances in fluids andgases. Many of these rely on specific ligand/antiligand reactions as themechanism of detection. That is, pairs of substances (i.e. the bindingpairs or ligand/antiligands) are known to bind to each other, whilebinding little or not at all to other substances. This has been thefocus of a number of techniques that utilize these binding pairs for thedetection of the complexes. These generally are done by labeling onecomponent of the complex in some way, so as to make the entire complexdetectable, using, for example, radioisotopes, fluorescent and otheroptically active molecules, enzymes, etc.

Other assays rely on electronic signals for detection. Of particularinterest are biosensors. At least two types of biosensors are known;enzyme-based or metabolic biosensors and binding or bioaffinity sensors.See for example U.S. Pat. Nos. 4,713,347; 5,192,507; 4,920,047;3,873,267; and references disclosed therein. While some of these knownsensors use alternating current (AC) techniques, these techniques aregenerally limited to the detection of differences in bulk (ordielectric) impedance, and rely on the use of mediators in solution toshuttle the charge to the electrode.

Recent work utilizes electron transfer for detection of nucleic acid andother analytes. See U.S. Pat. Nos. 5,824,473; 5,770,369; 5,591,578;5,705,348; 5,780,234; PCT US98/12430; PCT US97/20014; PCT US95/14621;and PCT US98/12082.

The formation of specific protein-ligand complexes is often accompaniedby large conformational changes (Uversky et al., Biochemistry (Moscow),63:420-433, 1998), up to and including the ligand-induced folding ofproteins unfolded under physiological conditions (reviewed in Plaxco andGross, Nature, 386:657-659, 1997). In addition, a number of studiessuggest that via deletions (e.g. Hamill et al., Biochemistry,37:8071-8079, 1998; Flanagan et al., Proc. Natl. Acad. Sci. USA,89:748-52, 1992) or core simplification (reviewed in Plaxco et al.,Curr. Op. Struct. Biol., 8:80-85,1998; Desjarlis and Handel Prot. Sci.,4:2006-2018, 1995; Gassner et al., Proc. Natl. Acad. Sci. USA,93:12155-12158, 1996) it may be possible to rationally engineerligand-induced folding into naturally occurring or pahge-displaygenerated (e.g. Vaughan et al., Nat. Biotechnol., 16:535-539, 1998;Wilson and Finlay, Can. J. Microbiol., 44:313-329, 1998) proteinsfeaturing any arbitrary ligand specificity. These results suggest thatan ability to conveniently monitor protein conformational changes couldprovide an important and commerically viable means of monitoring a widevariety of ligands of significant clinical, industrial, environmental ormilitary interest.

While ligand-induced conformational changes could serve as an indicatorof the presence or many specific ligands, the cumbersome, costlycrystallographic or spectroscopic (reviewed in Plaxco and Dobson, Curr.Opin. Struc. Biol., 630-636, 1996) equipment required to monitor proteinconformational changes has precluded the development of a viableligand-detection technique based on this approach.

Electron transfer rates through biopolymers are extremely sensitive todonor-acceptor geometry, the structure and conformation of theintervening medium and the relative free and reorganization energies ofthe redox centers; see Casimiro et al., J. Phys. Chem., 97:13073-13077,1993; Dutton and Mosser, Proc. Natl. Acad. Sci. USA, 91:10247-10250,1994; Bjerrum et al., J. Bioenerg. Biomembr., 27:295-302, 1995; Langenet al., Science, 268:1733-1735, 1995; Daizadeh et al., Proc. Natl. Acad.Sci. USA, 94:3703-3708, 1998). In addition, the effects of proteinconformational change on through-protein electron transfer rates hasbeen reported; see Mutz et al., Proc. Natl. Acad. Sci. USA,93:9521-9526,1996.

Accordingly, it is an object of the invention to provide for biosensorsthat can detect conformational changes that occur in proteins uponbinding of ligands on the basis of electron transfer.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A, 1B and 1C schematically depict several embodiments of theinvention. FIG. 1A shows attachment to electrode 50 of binding ligand100 via an attachment linker (depicted as a conductive oligomer 60,although insulators 55 can be used as well). Upon binding of the ligand30, the binding ligand undergoes a conformational change that allowselectron transfer between the ETM 10 and the electrode 50 via theconductive oligomer 60. FIG. 1B depicts the solution system, comprisingtwo ETMs 10 and 15, one of which is an electron donor and the other isan acceptor. Similarly, FIG. 1C depicts a direct attachment of thebinding ligand to the electrode.

FIG. 2 depicts the well-characterized structure and thermodynamics ofthe hin-hix complex. The two naturally occurring histidine residues(represented with side-chains) residues in the protein and thecarboxy-terminal six residues of the domain provide several ETM sites.Previous studies indicate that the carboxy-terminal tail of hin remainsunstructured in the complex; this terminus is thus a good site forattachment to a surface such as an electrode as is outlined herein.

FIG. 3 depicts a fmoc protected arylthiol conductive oligomer. SeeCreager et al., Analytica Chem, in Press, 1999; Yu et al., AnalyticaChem., Submitted, 1999.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is based on previous work that indicates thatelectron transfer serves as an effective detection method for targetanalytes, including nucleic acids. See U.S. Pat. Nos. 5,824,473;5,770,369; 5,591,578; 5,705,348; 5,780,234; PCT US98/12430; PCTUS97/20014; PCT US95/14621; and PCT US98/12082; and U.S. Ser. Nos.09/134,058; 08/911,589; 09/135,183; 60/084,652; 60/084,425; and60/105,875, all of which are hereby expressly incorporated by referencein their entirety.

Accordingly, the present invention provides methods of detecting thepresence of target analytes. By “target analyte” or “analyte” or“ligand” or grammatical equivalents herein is meant any molecule,compound or particle to be detected and that causes a conformationalchange in a binding ligand as is more fully outlined below. As will beappreciated by those in the art, a large number of analytes may bedetected using the present methods.

Suitable analytes include organic and inorganic molecules, includingbiomolecules. In a preferred embodiment, the analyte may be anenvironmental pollutant (including pesticides, insecticides, toxins,etc.); a chemical (including solvents, polymers, organic materials,etc.); therapeutic molecules (including therapeutic and abused drugs,antibiotics, etc.); biomolecules (including hormones, cytokines,proteins, lipids, carbohydrates, cellular membrane antigens andreceptors (neural, hormonal, nutrient, and cell surface receptors) ortheir ligands, etc); whole cells (including procaryotic (such aspathogenic bacteria) and eukaryotic cells, including mammalian tumorcells); viruses (including retroviruses, herpesviruses, adenoviruses,lentiviruses, etc.); and spores; etc. Particularly preferred analytesare environmental pollutants; proteins (including enzymes, antibodies,antigens, growth factors, cytokines, etc); therapeutic and abused drugs;cells; and viruses.

In a preferred embodiment, the target analyte is a protein. As will beappreciated by those in the art, there are a large number of possibleproteinaceous tarot analytes that may be detected using the presentinvention. By “proteins” or grammatical equivalents herein is meantproteins, oligopeptides and peptides, derivatives and analogs, includingproteins containing non-naturally occurring amino acids and amino acidanalogs, and peptidomimetic structures. The side chains may be in eitherthe (R) or the (S) configuration. In a preferred embodiment, the aminoacids are in the (S) or L-configuration. As discussed below, when theprotein is used as a binding ligand, it may be desirable to utilizeprotein analogs to retard degradation by sample contaminants.

Suitable protein target analytes include, but are not limited to, (1)immunoglobulins, particularly IgEs, IgGs and IgMs, and particularlytherapeutically or diagnostically relevant antibodies, including but notlimited to, for example, antibodies to human albumin, apolipoproteins(including apolipoprotein E), human chorionic gonadotropin, cortisol,a-fetoprotein, thyroxin, thyroid stimulating hormone (TSH),antithrombin, antibodies to pharmaceuticals (including antieptilepticdrugs (phenytoin, primidone, carbariezepin, ethosuximide, valproic acid,and phenobarbitol), cardioactive drugs (digoxin, lidocaine,procainamide, and disopyramide), bronchodilators (theophylline),antibiotics (chloramphenicol, sulfonamides), antidepressants,immunosuppresants, abused drugs (amphetamine, methamphetamine,cannabinoids, cocaine and opiates) and antibodies to any number ofviruses (including orthomyxoviruses, (e.g. influenza virus),paramyxoviruses (e.g respiratory syncytial virus, mumps virus, measlesvirus), adenoviruses, rhinoviruses, coronaviruses, reoviruses,togaviruses (e.g. rubella virus), parvoviruses, poxviruses (e.g. variolavirus, vaccinia virus), enteroviruses (e.g. poliovirus, coxsackievirus),hepatitis viruses (including A, B and C), herpesviruses (e.g. Herpessimplex virus, varicella-zoster virus, cytomegalovirus, Epstein-Barrvirus), rotaviruses, Norwalk viruses, hantavirus, arenavirus,rhabdovirus (e.g. rabies virus), retroviruses (including HIV, HTLV-I and-II), papovaviruses (e.g. papillomavirus), polyomaviruses, andpicornaviruses, and the like), and bacteria (including a wide variety ofpathogenic and non-pathogenic prokaryotes of interest includingBacillus; Vibrio, e.g. V. cholerae; Escherichia, e.g. Enterotoxigenic E.coli, Shigella, e.g. S. dysenteriae; Salmonella, e.g. S. typhi;Mycobacterium e.g. M. tuberculosis, M. leprae; Clostridium, e.g. C.botulinum, C. tetani, C. difficile, C.perfringens; Cornyebacterium, e.g.C. diphtheriae; Streptococcus, S. pyogenes, S. pneumoniae;Staphylococcus, e.g. S. aureus; Haemophilus, e.g. H. influenzae;Neisseria, e.g. N. meningitidis, N. gonorrhoeae; Yersinia, e.g. G.lamblia Y. pestis, Pseudomonas, e.g. P. aeruginosa, P. putida;Chlamydia, e.g. C. trachomatis; Bordetella, e.g. B. pertussis;Treponema, e.g. T. palladium; and the like); (2) enzymes (and otherproteins), including but not limited to, enzymes used as indicators ofor treatment for heart disease, including creatine kinase, lactatedehydrogenase, aspartate amino transferase, troponin T, myoglobin,fibrinogen, cholesterol, triglycerides, thrombin, tissue plasminogenactivator (tPA); pancreatic disease indicators including amylase,lipase, chymotrypsin and trypsin; liver function enzymes and proteinsincluding cholinesterase, bilirubin, and alkaline phosphotase; aldolase,prostatic acid phosphatase, terminal deoxynucleotidyl transferase, andbacterial and viral enzymes such as HIV protease; (3) hormones andcytokines (many of which serve as ligands for cellular receptors) suchas erythropoietin (EPO), thrombopoietin (TPO), the interleukins(including IL-1 through IL-17), insulin, insulin-like growth factors(including IGF-1 and -2), epidermal growth factor (EGF), transforminggrowth factors (including TGF-α and TGF-β), human growth hormone,transferrin, epidermal growth factor (EGF), low density lipoprotein,high density lipoprotein, leptin, VEGF, PDGF, ciliary neurotrophicfactor, prolactin, adrenocorticotropic hormone (ACTH), calcitonin, humanchorionic gonadotropin, cotrisol, estradiol, follicle stimulatinghormone (FSH), thyroid-stimulating hormone (TSH), leutinzing hormone(LH), progeterone, testosterone; and (4) other proteins (includingα-fetoprotein, carcinoembryonic antigen CEA.

In addition, any of the biomolecules for which antibodies may bedetected may be detected directly as well; that is, detection of virusor bacterial cells, proteins, enzymes, therapeutic and abused drugs,etc., may be done directly.

Suitable target analytes include carbohydrates, including but notlimited to, markers for breast cancer (CA15-3, CA 549, CA 27.29),mucin-like carcinoma associated antigen (MCA), ovarian cancer (CA125),pancreatic cancer (DE-PAN-2), and colorectal and pancreatic cancer (CA19, CA 50, CA242).

Suitable target analytes include metal ions, particularly heavy and/ortoxic metals, including but not limited to, aluminum, arsenic, cadmium,selenium, cobalt, copper, chromium, lead, silver and nickel.

A sample containing the target analyte is added to a compositioncomprising a binding ligand. By “binding ligand” or grammaticalequivalents herein is meant a compound that is used to probe for thepresence of the target analyte, and that will undergo a conformationalchange upon binding of the analyte.

As will be appreciated by those in the art, the composition of thebinding ligand will depend on the composition of the target analyte.Binding ligands for a wide variety of analytes are known or can bereadily found using known techniques. For example, when the analyte is aprotein, the binding ligands include proteins (particularly includingantibodies or fragments thereof (FAbs, etc.)) or small molecules. Whenthe analyte is a metal ion, the binding ligand generally comprisestraditional metal ion ligands or chelators which causes the bindingligand to undergo a conformational change as a result of binding of themetal ion. Preferred binding ligand proteins include peptides. Forexample, when the analyte is an enzyme, suitable binding ligands includesubstrates and inhibitors. Antigen-antibody pairs, receptor-ligands, andcarbohydrates and their binding partners are also suitableanalyte-binding ligand pairs if an appropriate conformational change canoccur. The binding ligand may be nucleic acid, when nucleic acid bindingproteins are the targets; alternatively, as is generally described inU.S. Pat. Nos. 5,270,163, 5,475,096, 5,567,588, 5,595,877, 5,637,459,5,683,867, 5,705,337, and related patents, hereby incorporated byreference, nucleic acid “aptomers” can be developed for binding tovirtually any target analyte. Similarly, there is a wide body ofliterature relating to the development of binding partners based oncombinatorial chemistry methods. In this embodiment, when the bindingligand is a nucleic acid, preferred compositions and techniques areoutlined in PCT US97/20014, hereby incorporated by reference.

By “nucleic acid” or “oligonucleotide” or grammatical equivalents hereinmeans at least two nucleotides covalently linked together. A nucleicacid of the present invention will generally contain phosphodiesterbonds, although in some cases, as outlined below, nucleic acid analogsare included that may have alternate backbones, comprising, for example,phosphoramide (Beaucage et al., Tetrahedron 49(10):1925 (1993) andreferences therein; Letsinger, J. Org. Chem. 35:3800 (1970); Sprinzl etal., Eur. J. Biochem. 81:579 (1977); Letsinger et al., Nucl. Acids Res.14:3487 (1986); Sawai et al., Chem. Lett. 805 (1984), Letsinger et al.,J. Am. Chem. Soc. 110:4470 (1988); and Pauwels et al., Chemica Scripta26:141 91986)), phosphorothioate (Mag et al., Nucleic Acids Res. 19:1437(1991); and U.S. Pat. No. 5,644,048), phosphorodithioate (Briu et al.,J. Am. Chem. Soc. 111:2321 (1989), O-methylphophoroamidite linkages (seeEckstein, Oligonucleotides and Analogues: A Practical Approach, OxfordUniversity Press), and peptide nucleic acid backbones and linkages (seeEgholm, J. Am. Chem. Soc. 114:1895 (1992); Meier et al., Chem. Int. Ed.Engl. 31:1008 (1992); Nielsen, Nature, 365:566 (1993); Carlsson et al.,Nature 380:207 (1996), all of which are incorporated by reference).Other analog nucleic acids include those with positive backbones (Denpcyet al., Proc. Natl. Acad. Sci. USA 92:6097 (1995); non-ionic backbones(U.S. Pat. Nos. 5,386,023, 5,637,684, 5,602,240, 5,216,141 and4,469,863; Kiedrowshi et al., Angew. Chem. Intl. Ed. English 30:423(1991); Letsinger et al., J. Am. Chem. Soc. 110:4470 (1988); Letsingeret al., Nucleoside & Nucleotide 13:1597 (1994); Chapters 2 and 3, ASCSymposium Series 580, “Carbohydrate Modifications in AntisenseResearch”. Ed. Y. S. Sanghui and P. Dan Cook; Mesmaeker et al.,Bioorganic & Medicinal Chem. Lett. 4:395 (1994); Jeffs et al., J.Biomolecular NMR 34:17 (1994); Tetrahedron Lett. 37:743 (1996)) andnon-ribose backbones, including those described in U.S. Pat. Nos.5,235,033 and 5,034,506, and Chapters 6 and 7, ASC Symposium Series 580,“Carbohydrate Modifications in Antisense Research”, Ed. Y. S. Sanghuiand P. Dan Cook. Nucleic acids containing one or more carbocyclic sugarsare also included within the definition of nucleic acids (see Jenkins etal., Chem. Soc. Rev. (1995) pp169-176). Several nucleic acid analogs aredescribed in Rawls, C & E News Jun. 2, 1997 page 35. All of thesereferences are hereby expressly incorporated by reference. Thesemodifications of the ribose-phosphate backbone may be done to facilitatethe addition of electron transfer moieties (ETMs) or conductiveoligomers, or to increase the stability and half-life of such moleculesin physiological environments.

As will be appreciated by those in the art, all of these nucleic acidanalogs may find use in the present invention. In addition, mixtures ofnaturally occurring nucleic acids and analogs can be made; for example,at the site of conductive oligomer or ETM attachment, an analogstructure may be used. Alternatively, mixtures of different nucleic acidanalogs, and mixtures of naturally occuring nucleic acids and analogsmay be made.

In a preferred embodiment, the binding of the target analyte to thebinding ligand is specific, and the binding ligand is part of a bindingpair. By “specifically bind” herein is meant that the ligand binds theanalyte, with specificity sufficient to differentiate between theanalyte and other components or contaminants of the test sample.However, as will be appreciated by those in the art, it will be possibleto detect analytes using binding which is not highly specific; forexample, the systems may use different binding ligands, for example anarray of different ligands, and detection of any particular analyte isvia its “signature” of binding to a panel of binding ligands, similar tothe manner in which “electronic noses” work. This finds particularutility in the detection of chemical analytes. The binding should besufficient to remain bound under the conditions of the assay, includingwash steps to remove non-specific binding. In some embodiments, forexample in the detection of certain biomolecules, the disassociationconstants of the analyte to the binding ligand will be less than about10⁻⁴-10⁻⁶ M⁻¹, with less than about 10⁻⁵ to 10⁻⁹ M³¹ ¹ being preferredand less than about 10⁻⁷-10⁻¹ being particularly preferred.

As will be appreciated by those in the art, the binding ligand can benaturally occurring or a recombinant, designed binding ligand. Forexample, there are a number of proteins known to undergo conformationalchanges upon ligand binding that can be engineered for altered ligandspecificities. For example, there are a large number of ligand/receptorpairs that result in receptor dimerization upon ligand binding, such asa number of cell surface receptors including, but are not limited to,insulin receptor, insulin-like growth factor receptor, growth hormonereceptor, glucose transporters (particularly GLUT 4 receptor),transferrin receptor, epidermal growth factor receptor, low densitylipoprotein receptor, high density lipoprotein receptor, epidermalgrowth factor receptor, leptin receptor, interleukin receptors includingIL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-11, IL-12,IL-13, IL-15, and IL-17 receptors, human growth hormone receptor, VEGFreceptor, PDGF receptor, EPO receptor, TPO receptor, ciliaryneurotrophic factor receptor, prolactin receptor, and T-cell receptors.The engineering of two ligand binding sites connected by a flexiblelinker can result in a suitable binding ligand/target analyte pair.

In addition, there are a variety of known binding ligands that undergoconformational changes upon target analyte binding, that can beengineered to contain altered ligand specificity. Calmodulin, forexample, is the subject of extensive efforts to generate a variantphage-display library featuring a broad spectrum of ligandspecificities.

In a preferred embodiment, the target analyte and the binding ligand arenot both nucleic acid.

The target analyte and the binding ligand interact to form an assaycomplex. “Assay complex” herein is meant the collection of targetanalytes and binding ligands that contains at least one ETM and thusallows detection. The composition of the assay complex can vary, assandwich assays (i.e. more than one binding ligand used) can be run, aswill be appreciated by those in the art.

The binding ligand further comprises at least one electron transfermoiety (ETM). By “redox active molecule” or “RAM” or “electron transfermoiety”or “ETM” herein is meant a compound which is capable ofreversibly, semi-reversibly, or irreversibly transferring one or moreelectrons. The terms “electron donor moiety”, “electron acceptormoiety”, and “electron transfer moieties” or grammatical equivalentsherein refers to molecules capable of electron transfer under certainconditions. It is to be understood that electron donor and acceptorcapabilities are relative; that is, a molecule which can lose anelectron under certain experimental conditions will be able to accept anelectron under different experimental conditions. It is to be understoodthat the number of possible electron donor moieties and electronacceptor moieties is very large, and that one skilled in the art ofelectron transfer compounds will be able to utilize a number ofcompounds in the present invention. Preferred electron transfer moietiesinclude, but are not limited to, transition metal complexes, organicelectron transfer moieties, and electrodes.

In a preferred embodiment, the electron transfer moieties are transitionmetal complexes. Transition metals include those whose atoms have apartial or complete d shell of electrons; elements having the atomicnumbers 21-30, 39-48, 57-80 and the lanthanide series. Suitabletransition metals for use in the invention include, but are not limitedto, cadmium (Cd), copper (Cu), cobalt (Co), palladium (Pd), zinc (Zn),iron (Fe), ruthenium (Ru), rhodium (Rh), osmium (Os), rhenium (Re),platinium (Pt), scandium (Sc), titanium (Ti), Vanadium (V), chromium(Cr), manganese (Mn), nickel (Ni), Molybdenum (Mo), technetium (Tc),tungsten (W), and iridium (Ir). That is, the first series of transitionmetals, the platinum metals (Ru, Rh, Pd, Os, Ir and Pt), along with Fe,Re, W, Mo and Tc, are preferred. Particularly preferred are ruthenium,rhenium, osmium, platinium, cobalt and iron.

The transition metals are complexed with a variety of ligands, generallydepicted herein as “L”, to form suitable transition metal complexes, asis well known in the art. Suitable ligands fall into two categories:ligands which use nitrogen, oxygen, sulfur, carbon or phosphorus atoms(depending on the metal ion) as the coordination atoms (generallyreferred to in the literature as sigma (σ) donors) and organometallicligands such as metallocene ligands (generally referred to in theliterature as pi (π) donors, and depicted herein as L_(m)). Suitablenitrogen donating ligands are well known in the art and include, but arenot limited to, NH₂; NHR; NRR′; pyridine; pyrazine; isonicotinamide;imidazole; bipyridine and substituted derivatives of bipyridine;terpyridine and substituted derivatives; phenanthrolines, particularly1,10-phenanthroline (abbreviated phen) and substituted derivatives ofphenanthrolines such as 4,7-dimethylphenanthroline anddipyridol[3,2-a:2′, 3′-c]phenazine (abbreviated dppz);dipyridophenazine; 1,4,5,8,9,12-hexaazatriphenylene (abbreviated hat);9,10-phenanthrenequinone diimine (abbreviated phi);1,4,5,8-tetraazaphenanthrene (abbreviated tap);1,4,8,11-tetra-azacyclotetradecane (abbreviated cyclam) and isocyanide.Substituted derivatives, including fused derivatives, may also be used.In some embodiments, porphyrins and substituted derivatives of theporphyrin family may be used. See for example, ComprehensiveCoordination Chemistry, Ed. Wilkinson et al., Pergammon Press, 1987,Chapters 13.2 (pp73-98), 21.1 (pp. 813-898) and 21.3 (pp 915-957), allof which are hereby expressly incorporated by reference.

Suitable sigma donating ligands using carbon, oxygen, sulfur andphosphorus are known in the art. For example, suitable sigma carbondonors are found in Cotton and Wilkenson, Advanced Organic Chemistry,5th Edition, John Wiley & Sons, 1988, hereby incorporated by reference;see page 38, for example. Similarly, suitable oxygen ligands includecrown ethers, water and others known in the art. Phosphines andsubstituted phosphines are also suitable; see page 38 of Cotton andWilkenson.

The oxygen, sulfur, phosphorus and nitrogen-donating ligands areattached in such a manner as to allow the heteroatoms to serve ascoordination atoms.

In a preferred embodiment, organometallic ligands are used. In additionto purely organic compounds for use as redox moieties, and varioustransition metal coordination complexes with δ-bonded organic ligandwith donor atoms as heterocyclic or exocyclic substituents, there isavailable a wide variety of transition metal organometallic compoundswith π-bonded organic ligands (see Advanced Inorganic Chemistry, 5thEd., Cotton & Wilkinson, John Wiley & Sons, 1988, chapter 26;Organometallics, A Concise Introduction, Elschenbroich et al., 2nd Ed.,1992, VCH; and Comprehensive Organometallic Chemistry II, A Review ofthe Literature 1982-1994, Abel et al. Ed., Vol. 7, chapters 7, 8, 10 &11, Pergamon Press, hereby expressly incorporated by reference). Suchorganometallic ligands include cyclic aromatic compounds such as thecyclopentadienide ion [C₅H₅(−1)] and various ring substituted and ringfused derivatives, such as the indenylide (−1) ion, that yield a classof bis(cyclopentadieyl) metal compounds, (i.e. the metallocenes); seefor example Robins et al., J. Am. Chem. Soc. 104:1882-1893 (1982); andGassman et al., J. Am. Chem. Soc. 108:4228-4229(1986), incorporated byreference. Of these, ferrocene [(C₅H₅)₂Fe] and its derivatives areprototypical examples which have been used in a wide variety of chemical(Connelly et al., Chem. Rev. 96:877-910 (1996), incorporated byreference) and electrochemical (Geiger et al., Advances inOrganometallic Chemistry 23:1-93; and Geiger et al., Advances inOrganometallic Chemistry 24:87, incorporated by reference) electrontransfer or “redox” reactions. Metallocene derivatives of a variety ofthe first, second and third row transition metals are potentialcandidates as redox moieties. Other potentially suitable organometallicligands include cyclic arenes such as benzene, to yield bis(arene)metalcompounds and their ring substituted and ring fused derivatives, ofwhich bis(benzene)chromium is a prototypical example, Other acyclicπ-bonded ligands such as the allyl(−1) ion, or butadiene yieldpotentially suitable organometallic compounds, and all such ligands, inconjuction with other π-bonded and δ-bonded ligands constitute thegeneral class of organometallic compounds in which there is a metal tocarbon bond. Electrochemical studies of various dimers and oligomers ofsuch compounds with bridging organic ligands, and additionalnon-bridging ligands, as well as with and without metal-metal bonds arepotential candidate redox moieties.

As described herein, any combination of ligands may be used. Preferredcombinations include: a) all ligands are nitrogen donating ligands; b)all ligands are organometallic ligands; and c) the ligand at theterminus of the conductive oligomer is a metallocene ligand and theligand provided by the binding ligand is a nitrogen donating ligand,with the other ligands, if needed, are either nitrogen donating ligandsor metallocene ligands, or a mixture.

In addition, it may be desirable to use coordination sites of thetransition metal ion for attachment of the redox active molecule to abinding ligand (directly or indirectly using a linker). Thus forexample, when the ETM is directly joined to a binding ligand, one, twoor more of the coordination sites of the metal ion may be occupied bycoordination atoms supplied by the binding ligand (or by the linker, ifindirectly joined).

In addition to transition metal complexes, other organic electron donorsand acceptors may be used in the invention. These organic moleculesinclude, but are not limited to, riboflavin, xanthene dyes, azine dyes,acridine orange, N,N′-dimethyl-2,7-diazapyrenium dichloride (DAP²⁺),methylviologen, ethidium bromide, quinones such asN,N′-dimethylanthra(2,1,9-def:6,5,10-d′e′f′)diisoquinoline dichloride(ADIQ²⁺); porphyrins ([meso-tetrakis(N-methyl-x-pyridinium)porphyrintetrachloride], varlamine blue B hydrochloride, Bindschedler's green;2,6-dichloroindophenol, 2,6-dibromophenolindophenol; Brilliant crestblue (3-amino-9-dimethyl-amino-10-methylphenoxyazine chloride),methylene blue; Nile blue A (aminoaphthodiethylaminophenoxazinesulfate), indigo-5,5′,7,7′-tetrasulfonic acid, indigo-5,5′,7-trisulfonicacid; phenosafranine, indigo-5-monosulfonic acid; safranine T;bis(dimethylglyoximato)-iron(II) chloride; induline scarlet, neutralred, anthracene, coronene, pyrene, 9-phenylanthracene, rubrene,binaphthyl, DPA, phenothiazene, fluoranthene, phenanthrene, chrysene,1,8-diphenyl-1,3,5,7-octatetracene, naphthalene, acenaphthalene,perylene, TMPD and analogs and subsitituted derivatives of thesecompounds.

In one embodiment, the electron donors and acceptors are redox proteinsas are known in the art. However, redox proteins in many embodiments arenot preferred.

The choice of the specific electron transfer moieties will be influencedby the type of electron transfer detection used, as is generallyoutlined below.

As will be appreciated by those in the art, the present invention can beutilized in a number of ways as is generally depicted in FIG. 1. In apreferred embodiment, at least one electron donor moiety and at leastone electron acceptor moiety are attached, preferably covalently, to abinding ligand, such that upon binding of the target analyte, aconformational change occurs in the binding ligand and the donor andacceptor are brought into proximity, resulting in an increase in therate of electron transfer. This may be done in solution.

Alternatively, in a preferred embodiment, the binding ligand is attachedto an electrode, and comprises at least one ETM. As a result of targetanalyte binding, the ETM is brought into proximity of the electrode,either a bare electrode or an electrode comprising passivation agents,preferably conductive oligomers, such that electron transfer occursbetween the electrode and the ETM.

In a preferred embodiment, the binding ligand comprises at least oneelectron donor and at least one electron acceptor moiety. In general,the ETMs are attached to the binding ligands in a variety of ways, as isgenerally described in U.S. Pat. Nos. 5,824,473; 5,770,369; 5,591,578;5,705,348; 5,780,234; PCT US98/12430; PCT US97/20014; PCT US95/14621;and PCT US98/12082; and U.S. Ser. Nos. 09/134,058; 08/911,589;09/135,183; 60/084,652; 60/105,875; and 60/084,652, all of which arehereby expressly incorporated by reference in their entirety. In apreferred embodiment, the attachment is covalent.

A preferred embodiment utilizes proteinaceous binding ligands. As isknown in the art, any number of techniques may be used to attach an ETMto a proteinaceous binding ligand. A wide variety of techniques areknown to add moieties to proteins; a preferred embodiment utilizescoordination atoms of the amino acid side chains to bind transitionmetal complexes. Alternatively, functional groups on the ETMs and/orbinding ligands can be used for the covalent attachment of ETMs.Preferred functional groups for attachment are amino groups, carboxygroups, oxo groups and thiol groups, with amino groups beingparticularly preferred. These functional groups can then be used fordirect attachment of the moieties, or can be used for indirectattachment via linkers. Suitable linkers include, but are not limitedto, alkyl linkers (including heteroalkyl (including (poly)ethyleneglycol-type structures), substituted alkyl, aryalkyl linkers, etc. Asabove, the linkers will comprise one or more functional groups for theattachment of ETMs, which will be done as will be appreciated by thosein the art, for example through the use homo-or hetero-bifunctionallinkers as are well known (see 1994 Pierce Chemical Company catalog,technical section on cross-linkers, pages 155-200, incorporated hereinby reference).

A preferred embodiment utilizes nucleic acids as the binding ligand,with techniques outlined in PCT US97/20014 being useful for attachment.

In a preferred embodiment, a plurality of ETMs can be covalentlyattached to a binding ligand as is described in U.S. Pat. Nos.5,824,473; 5,770,369; 5,591,578; 5,705,348; 5,780,234; PCT US98/12430;PCT US97/20014; PCT US95/14621; and PCT US98/12082; and U.S. Ser. Nos.09/134,058; 08/911,589; 09/135,183; 60/084,652; 60/084,425; and60/105,875, all of which are hereby expressly incorporated by referencein their entirety.

In a preferred embodiment, at least one ETM is covalently attached tothe binding ligand as outlined above, and the binding ligand iscovalently attached to an electrode. By “electrode” herein is meant acomposition, which, when connected to an electronic device, is able tosense a current or charge and convert it to a signal. Alternatively anelectrode can be defined as a composition which can apply a potential toand/or pass electrons to or from species in the solution. Thus, anelectrode is an ETM as described herein. Preferred electrodes are knownin the art and include, but are not limited to, certain metals and theiroxides, including gold; platinum; palladium; silicon; aluminum; metaloxide electrodes including platinum oxide, titanium oxide, tin oxide,indium tin oxide, palladium oxide, silicon oxide, aluminum oxide,molybdenum oxide (Mo₂O₆), tungsten oxide (WO₃) and ruthenium oxides; andcarbon (including glassy carbon electrodes, graphite and carbon paste).Preferred electrodes include gold, silicon, carbon and metal oxideelectrodes, with gold being particularly preferred.

The electrodes described herein are depicted as a flat surface, which isonly one of the possible conformations of the electrode and is forschematic purposes only. The conformation of the electrode will varywith the detection method used. For example, flat planar electrodes maybe preferred for certain detection methods, or when arrays of bindingligands are made, thus requiring addressable locations for bothsynthesis and detection. Alternatively, for single probe analysis, theelectrode may be in the form of a tube, with the SAMs comprisingconductive oligomers and binding ligands bound to the inner surface.This allows a maximum of surface area containing the binding ligands tobe exposed to a small volume of sample.

In a preferred embodiment, the electrode further comprises a passivationagent, preferably in the form of a monolayer on the electrode surface.Passivation agents include conductive oligomers and insulators, asdefined below. By “monolayer” or “self-assembled monolayer” or “SAM”herein is meant a relatively ordered assembly of molecules spontaneouslychemisorbed on a surface, in which the molecules are orientedapproximately parallel to each other and roughly perpendicular to thesurface. Each of the molecules includes a functional group that adheresto the surface, and a portion that interacts with neighboring moleculesin the monolayer to form the relatively ordered array. A “mixed”monolayer comprises a heterogeneous monolayer, that is, where at leasttwo different molecules make up the monolayer. The SAM may compriseconductive oligomers alone, or a mixture of conductive oligomers andinsulators. As outlined herein, the efficiency of target analyte bindingmay increase when the binding ligand is at a distance from theelectrode. Similarly, non-specific binding of biomolecules to anelectrode is generally reduced when a monolayer is present. Thus, amonolayer facilitates the maintenance of the binding ligand away fromthe electrode surface. In addition, a monolayer serves to keep chargecarriers away from the surface of the electrode. Thus, this layer helpsto prevent electrical contact between the electrodes and the ETMs, orbetween the electrode and charged species within the solvent. Suchcontact can result in a direct “short circuit” or an indirect shortcircuit via charged species which may be present in the sample.Accordingly, the monolayer is preferably tightly packed in a uniformlayer on the electrode surface, such that a minimum of “holes” exist.The monolayer thus serves as a physical barrier to block solventaccesibility to the electrode.

In a preferred embodiment, the monolayer comprises conductive oligomers.By “conductive oligomer” herein is meant a substantially conductingoligomer, preferably linear, some embodiments of which are referred toin the literature as “molecular wires”. By “substantially conducting”herein is meant that the oligomer is capable of transfering electrons at100 Hz. Generally, the conductive oligomer has substantially overlappingπ-orbitals, i.e. conjugated π-orbitals, as between the monomeric unitsof the conductive oligomer, although the conductive oligomer may alsocontain one or more sigma (σ) bonds. Additionally, a conductive oligomermay be defined functionally by its ability to inject or receiveelectrons into or from an associated ETM. Furthermore, the conductiveoligomer is more conductive than the insulators as defined herein.Additionally, the conductive oligomers of the invention are to bedistinguished from electroactive polymers, that themselves may donate oraccept electrons.

In a preferred embodiment, the conductive oligomers have a conductivity,S, of from between about 10⁻⁶ to about 10⁴ Ω⁻¹cm⁻¹, with from about 10⁻⁵to about 10³ Ω⁻¹cm⁻¹ being preferred, with these S values beingcalculated for molecules ranging from about 20 Å to about 200 Å. Asdescribed below, insulators have a conductivity S of about 10⁻⁷ Ω⁻¹cm⁻¹or lower, with less than about 10 ⁻⁸ Ω⁻¹cm⁻¹ being preferred. Seegenerally Gardner et al., Sensors and Actuators A 51 (1995) 57-66,incorporated herein by reference.

Desired characteristics of a conductive oligomer include highconductivity, sufficient solubility in organic solvents and/or water forsynthesis and use of the compositions of the invention, and preferablychemical resistance to reactions that occur i) during binding ligandsynthesis (such that monomers containing the conductive oligomers may beadded to a synthesizer during the synthesis of the compositions of theinvention), ii) during the attachment of the conductive oligomer to anelectrode, or iii) during assays. In addition, conductive oligomers thatwill promote the formation of self-assembled monolayers are preferred.

The oligomers of the invention comprise at least two monomeric subunits,as described herein. As is described more fully below, oligomers includehomo- and hetero-oligomers, and include polymers.

In a preferred embodiment, the conductive oligomer has the structuredepicted in Structure 1:

As will be understood by those in the art, all of the structuresdepicted herein may have additional atoms or structures; i.e. theconductive oligomer of Structure 1 may be attached to ETMs, such aselectrodes, transition metal complexes, organic ETMs, and metallocenes,and to nucleic acids, or to several of these. Unless otherwise noted,the conductive oligomers depicted herein will be attached at the leftside to an electrode; that is, as depicted in Structure 1, the left “Y”is connected to the electrode as described herein. If the conductiveoligomer is to be attached to a nucleic acid, the right “Y”, if present,is attached to the nucleic acid, either directly or through the use of alinker, as is described herein.

In this embodiment, Y is an aromatic group, n is an integer from 1 to50, g is either 1 or zero, e is an integer from zero to 10, and m iszero or 1. When g is 1, B—D is a conjugated bond, preferably selectedfrom acetylene, alkene, substituted alkene, amide, azo, —C═N— (including—N═C—, —CR═N— and —N═CR—), —Si═Si—, and —Si═C— (including —C═Si—,—Si═CR— and —CR═Si—). When g is zero, e is preferably 1, D is preferablycarbonyl, or a heteroatom moiety, wherein the heteroatom is selectedfrom oxygen, sulfur, nitrogen, silicon or phosphorus. Thus, suitableheteroatom moieties include, but are not limited to, —NH and —NR,wherein R is as defined herein; substituted sulfur; sulfonyl (—SO₂—)sulfoxide (—SO—); phosphine oxide (—PO— and —RPO—); and thiophosphine(—PS— and —RPS—). However, when the conductive oligomer is to beattached to a gold electrode, as outlined below, sulfur derivatives arenot preferred.

By “aromatic group” or grammatical equivalents herein is meant anaromatic monocyclic or polycyclic hydrocarbon moiety generallycontaining 5 to 14 carbon atoms (although larger polycyclic ringsstructures may be made) and any carbocylic ketone or thioketonederivative thereof, wherein the carbon atom with the free valence is amember of an aromatic ring. Aromatic groups include arylene groups andaromatic groups with more than two atoms removed. For the purposes ofthis application aromatic includes heterocycle. “Heterocycle” or“heteroaryl” means an aromatic group wherein 1 to 5 of the indicatedcarbon atoms are replaced by a heteroatom chosen from nitrogen, oxygen,sulfur, phosphorus, boron and silicon wherein the atom with the freevalence is a member of an aromatic ring, and any heterocyclic ketone andthioketone derivative thereof. Thus, heterocycle includes thienyl,furyl, pyrrolyl, pyrimidinyl, oxalyl, indolyl, purinyl, quinolyl,isoquinolyl, thiazolyl, imidozyl, etc.

Importantly, the Y aromatic groups of the conductive oligomer may bedifferent, i.e. the conductive oligomer may be a heterooligomer. Thatis, a conductive oligomer may comprise a oligomer of a single type of Ygroups, or of multiple types of Y groups.

The aromatic group may be substituted with a substitution group,generally depicted herein as R. R groups may be added as necessary toaffect the packing of the conductive oligomers, i.e. R groups may beused to alter the association of the oligomers in the monolayer. Rgroups may also be added to 1) alter the solubility of the oligomer orof compositions containing the oligomers; 2) alter the conjugation orelectrochemical potential of the system; and 3) alter the charge orcharacteristics at the surface of the monolayer.

In a preferred embodiment, when the conductive oligomer is greater thanthree subunits, R groups are preferred to increase solubility whensolution synthesis is done. However, the R groups, and their positions,are chosen to minimally effect the packing of the conductive oligomerson a surface, particularly within a monolayer, as described below. Ingeneral, only small R groups are used within the monolayer, with largerR groups generally above the surface of the monolayer. Thus for examplethe attachment of methyl groups to the portion of the conductiveoligomer within the monolayer to increase solubility is preferred, withattachment of longer alkoxy groups, for example, C3 to C10, ispreferably done above the monolayer surface. In general, for the systemsdescribed herein, this generally means that attachment of stericallysignificant R groups is not done on any of the first two or threeoligomer subunits, depending on the average length of the moleculesmaking up the monolayer.

Suitable R groups include, but are not limited to, hydrogen, alkyl,alcohol, aromatic, amino, amido, nitro, ethers, esters, aldehydes,sulfonyl, silicon moieties, halogens, sulfur containing moieties,phosphorus containing moieties, and ethylene glycols. In the structuresdepicted herein, R is hydrogen when the position is unsubstituted. Itshould be noted that some positions may allow two substitution groups, Rand R′, in which case the R and R′ groups may be either the same ordifferent.

By “alkyl group” or grammatical equivalents herein is meant a straightor branched chain alkyl group, with straight chain alkyl groups beingpreferred. If branched, it may be branched at one or more positions, andunless specified, at any position. The alkyl group may range from about1 to about 30 carbon atoms (C1-C30), with a preferred embodimentutilizing from about 1 to about 20 carbon atoms (C1-C20), with about C1through about C12 to about C15 being preferred, and C1 to C5 beingparticularly preferred, although in some embodiments the alkyl group maybe much larger. Also included within the definition of an alkyl groupare cycloalkyl groups such as C5 and C6 rings, and heterocyclic ringswith nitrogen, oxygen, sulfur or phosphorus. Alkyl also includesheteroalkyl, with heteroatoms of sulfur, oxygen, nitrogen, and siliconebeing preferred. Alkyl includes substituted alkyl groups. By“substituted alkyl group” herein is meant an alkyl group furthercomprising one or more substitution moieties “R”, as defined above.

By “amino groups” or grammatical equivalents herein is meant —NH₂, —NHRand —NR₂ groups, with R being as defined herein.

By “nitro group” herein is meant an —NO₂ group.

By “sulfur containing moieties” herein is meant compounds containingsulfur atoms, including but not limited to, thia-, thio- andsulfo-compounds, thiols (—SH and —SR), and sulfides (—RSR—). By“phosphorus containing moieties” herein is meant compounds containingphosphorus, including, but not limited to, phosphines and phosphates. By“silicon containing moieties” herein is meant compounds containingsilicon.

By “ether” herein is meant an —O—R group. Preferred ethers includealkoxy groups, with —O—(CH₂)₂CH₃ and —O—(CH₂)₄CH₃ being preferred.

By “ester” herein is meant a —COOR group.

By “halogen” herein is meant bromine, iodine, chlorine, or fluorine.Preferred substituted alkyls are partially or fully halogenated alkylssuch as CF₃, etc.

By “aldehyde” herein is meant —RCHO groups.

By “alcohol” herein is meant —OH groups, and alkyl alcohols —ROH.

By “amido” herein is meant —RCONH— or RCONR— groups.

By “ethylene glycol” or “(poly)ethylene glycol” herein is meant a—(O—CH₂—CH₂)_(n)— group, although each carbon atom of the ethylene groupmay also be singly or doubly substituted, i.e. —(O—CR₂—CR₂)_(n)—, with Ras described above. Ethylene glycol derivatives with other heteroatomsin place of oxygen (i.e. —(N—CH₂—CH₂)_(n)— or —(S—CH₂—CH₂)_(n)—, or withsubstitution groups) are also preferred.

Preferred substitution groups include but are not limited to, methyl,ethyl, propyl, alkoxy groups such as —O—(CH₂)₂CH₃ and —O—(CH₂)₄CH₃ andethylene glycol and derivatives thereof.

Preferred aromatic groups include, but are not limited to, phenyl,naphthyl, naphthalene, anthracene, phenanthroline, pyrole, pyridine,thiophene, porphyrins, and substituted derivatives of each of these,included fused ring derivatives.

In the conductive oligomers depicted herein, when g is 1, B—D is a bondlinking two atoms or chemical moieties. In a preferred embodiment, B—Dis a conjugated bond, containing overlapping or conjugated π-orbitals.

Preferred B—D bonds are selected from acetylene (—C≡C—, also calledalkyne or ethyne), alkene (—CH═CH—, also called ethylene), substitutedalkene (—CR═CR—, —CH═CR— and —CR═CH—), amide (—NH—CO— and —NR—CO— or—CO—NH— and —CO—NR—), azo (—N═N—), esters and thioesters (—CO—O—,—O—CO—, —CS—O— and —O—CS—) and other conjugated bonds such as (—CH═N—,—CR═N—, —N═CH— and —N═CR—), (—SiH═SiH—, —SiR═SiH—, —SiR═SiH—, and—SiR═SiR—), (—SiH═CH—, —SiR═CH—, —SiH═CR—, —SiR═CR—, —CH═SiH—, —CR═SiH—,—CH═SiR—, and —CR═SiR—). Particularly preferred B—D bonds are acetylene,alkene, amide, and substituted derivatives of these three, and azo.Especially preferred B—D bonds are acetylene, alkene and amide. Theoligomer components attached to double bonds may be in the trans or cisconformation, or mixtures. Thus, either B or D may include carbon,nitrogen or silicon. The substitution groups are as defined as above forR.

When g=0 in the Structure 1 conductive oligomer, e is preferably 1 andthe D moiety may be carbonyl or a heteroatom moiety as defined above.

As above for the Y rings, within any single conductive oligomer, the B—Dbonds (or D moieties, when g=0) may be all the same, or at least one maybe different. For example, when m is zero, the terminal B—D bond may bean amide bond, and the rest of the B—D bonds may be acetylene bonds.Generally, when amide bonds are present, as few amide bonds as possibleare preferable, but in some embodiments all the B—D bonds are amidebonds. Thus, as outlined above for the Y rings, one type of B—D bond maybe present in the conductive oligomer within a monolayer as describedbelow, and another type above the monolayer level, for example to givegreater flexibility for nucleic acid hybridization when the nucleic acidis attached via a conductive oligomer.

In the structures depicted herein, n is an integer from 1 to 50,although longer oligomers may also be used (see for example Schumm etal., Angew. Chem. Int. Ed. Engl. 1994 33(13):1360). Without being boundby theory, it appears that for efficient hybridization of nucleic acidson a surface, the hybridization should occur at a distance from thesurface, i.e. the kinetics of hybridization increase as a function ofthe distance from the surface, particularly for long oligonucleotides of200 to 300 basepairs. Accordingly, when a nucleic acid is attached via aconductive oligomer, as is more fully described below, the length of theconductive oligomer is such that the closest nucleotide of the nucleicacid is positioned from about 6 Å to about 100 Å (although distances ofup to 500 Å may be used) from the electrode surface, with from about 15Å to about 60 Å being preferred and from about 25 Å to about 60 Å alsobeing preferred. Accordingly, n will depend on the size of the aromaticgroup, but generally will be from about 1 to about 20, with from about 2to about 15 being preferred and from about 3 to about 10 beingespecially preferred.

In the structures depicted herein, m is either 0 or 1. That is, when mis 0, the conductive oligomer may terminate in the B—D bond or D moiety,i.e. the D atom is attached to the nucleic acid either directly or via alinker. In some embodiments, for example when the conductive oligomer isattached to a phosphate of the ribose-phosphate backbone of a nucleicacid, there may be additional atoms, such as a linker, attached betweenthe conductive oligomer and the nucleic acid. Additionally, as outlinedbelow, the D atom may be the nitrogen atom of the amino-modified ribose.Alternatively, when m is 1, the conductive oligomer may terminate in Y,an aromatic group, i.e. the aromatic group is attached to the nucleicacid or linker.

As will be appreciated by those in the art, a large number of possibleconductive oligomers may be utilized. These include conductive oligomersfalling within the Structure 1 and Structure 8 formulas, as well asother conductive oligomers, as are generally known in the art, includingfor example, compounds comprising fused aromatic rings or Teflon®-likeoligomers, such as —(CF₂)_(n)—, —(CHF)_(n)— and —(CFR)_(n)—. See forexample, Schumm et al., Angew. Chem. Intl. Ed. Engl. 33:1361(1994);Grosshenny et al., Platinum Metals Rev. 40(1):26-35 (1996); Tour,Chem. Rev. 96:537-553 (1996); Hsung et al., Organometallics 14:4808-4815(1995; and references cited therein, all of which are expresslyincorporated by reference.

Particularly preferred conductive oligomers of this embodiment aredepicted below:

Structure 2 is Structure 1 when g is 1. Preferred embodiments ofStructure 2 include: e is zero, Y is pyrole or substituted pyrole; e iszero, Y is thiophene or substituted thiophene; e is zero, Y is furan orsubstituted furan; e is zero, Y is phenyl or substituted phenyl; e iszero, Y is pyridine or substituted pyridine; e is 1, B—D is acetyleneand Y is phenyl or substituted phenyl (see Structure 4 below). Apreferred embodiment of Structure 2 is also when e is one, depicted asStructure 3 below:

Preferred embodiments of Structure 3 are: Y is phenyl or substitutedphenyl and B—D is azo; Y is phenyl or substituted phenyl and B—D isacetylene; Y is phenyl or substituted phenyl and B—D is alkene; Y ispyridine or substituted pyridine and B—D is acetylene; Y is thiophene orsubstituted thiophene and B—D is acetylene; Y is furan or substitutedfuran and B—D is acetylene; Y is thiophene or furan (or substitutedthiophene or furan) and B—D are alternating alkene and acetylene bonds.

Most of the structures depicted herein utilize a Structure 3 conductiveoligomer. However, any Structure 3 oligomers may be substituted with anyof the other structures depicted herein, i.e. Structure 1 or 8 oligomer,or other conducting oligomer, and the use of such Structure 3 depictionis not meant to limit the scope of the invention.

Particularly preferred embodiments of Structure 3 include Structures 4,5, 6 and 7, depicted below:

Particularly preferred embodiments of Structure 4 include: n is two, mis one, and R is hydrogen; n is three, m is zero, and R is hydrogen; andthe use of R groups to increase solubility.

When the B—D bond is an amide bond, as in Structure 5, the conductiveoligomers are pseudopeptide oligomers. Although the amide bond inStructure 5 is depicted with the carbonyl to the left, i.e. —CONH—, thereverse may also be used, i.e. —NHCO—. Particularly preferredembodiments of Structure 5 include: n is two, m is one, and R ishydrogen; n is three, m is zero, and R is hydrogen (in this embodiment,the terminal nitrogen (the D atom) may be the nitrogen of theamino-modified ribose); and the use of R groups to increase solubility.

Preferred embodiments of Structure 6 include the first n is two, secondn is one, m is zero, and all R groups are hydrogen, or the use of Rgroups to increase solubility.

Preferred embodiments of Structure 7 include: the first n is three, thesecond n is from 1-3, with m being either 0 or 1, and the use of Rgroups to increase solubility.

In a preferred embodiment, the conductive oligomer has the structuredepicted in Structure 8:

In this embodiment, C are carbon atoms, n is an integer from 1 to 50, mis 0 or 1, J is a heteroatom selected from the group consisting ofoxygen, nitrogen, silicon, phosphorus, sulfur, carbonyl or sulfoxide,and G is a bond selected from alkane, alkene or acetylene, such thattogether with the two carbon atoms the C—G—C group is an alkene(—CH═CH—), substituted alkene (—CR═CR—) or mixtures thereof (—CH═CR— or—CR═CH—), acetylene (—C≡C—), or alkane (—CR₂—CR₂—, with R being eitherhydrogen or a substitution group as described herein). The G bond ofeach subunit may be the same or different than the G bonds of othersubunits: that is, alternating oligomers of alkene and acetylene bondscould be used, etc. However, when G is an alkane bond, the number ofalkane bonds in the oligomer should be kept to a minimum, with about sixor less sigma bonds per conductive oligomer being preferred. Alkenebonds are preferred, and are generally depicted herein, although alkaneand acetylene bonds may be substituted in any structure or embodimentdescribed herein as will be appreciated by those in the art.

In some embodiments, for example when ETMs are not present, if m=0 thenat least one of the G bonds is not an alkane bond.

In a preferred embodiment, the m of Structure 8 is zero. In aparticularly preferred embodiment, m is zero and G is an alkene bond, asis depicted in Structure 9:

The alkene oligomer of structure 9, and others depicted herein, aregenerally depicted in the preferred trans configuration, althougholigomers of cis or mixtures of trans and cis may also be used. Asabove, R groups may be added to alter the packing of the compositions onan electrode, the hydrophilicity or hydrophobicity of the oligomer, andthe flexibility, i.e. the rotational, torsional or longitudinalflexibility of the oligomer. n is as defined above.

In a preferred embodiment, R is hydrogen, although R may be also alkylgroups and polyethylene glycols or derivatives.

In an alternative embodiment, the conductive oligomer may be a mixtureof different types of oligomers, for example of structures 1 and 8.

In addition, the terminus of at least some of the conductive oligomersin the monolayer are electronically exposed. By “electronically exposed”herein is meant that upon the placement of an ETM in close proximity tothe terminus, and after initiation with the appropriate signal, a signaldependent on the presence of the ETM may be detected. The conductiveoligomers may or may not have terminal groups. Thus, in a preferredembodiment, there is no additional terminal group, and the conductiveoligomer terminates with one of the groups depicted in Structures 1 to9; for example, a B—D bond such as an acetylene bond. Alternatively, ina preferred embodiment, a terminal group is added, sometimes depictedherein as “Q”. A terminal group may be used for several reasons; forexample, to contribute to the electronic availability of the conductiveoligomer for detection of ETMs, or to alter the surface of the SAM forother reasons, for example to prevent non-specific binding. For example,there may be negatively charged groups on the terminus to form anegatively charged surface such that when a postively charged bindingligand is repelled or prevented from lying down on the surface, tofacilitate binding. Preferred terminal groups include —NH₂, —OH, —COOH,and alkyl groups such as —CH₃, and (poly)alkyloxides such as(poly)ethylene glycol, with —OCH₂CH₂OH, —(OCH₂CH₂O)₂H, —(OCH₂CH₂O)₃H,and —(OCH₂CH₂O)₄H being preferred.

In one embodiment, it is possible to use mixtures of conductiveoligomers with different types of terminal groups. Thus, for example,some of the terminal groups may facilitate detection, and some mayprevent non-specific binding.

It will be appreciated that the monolayer may comprise differentconductive oligomer species, although preferably the different speciesare chosen such that a reasonably uniform SAM can be formed. Thus, forexample, when nucleic acids are covalently attached to the electrodeusing conductive oligomers, it is possible to have one type ofconductive oligomer used to attach the nucleic acid, and another typefunctioning to detect the ETM. Similarly, it may be desirable to havemixtures of different lengths of conductive oligomers in the monolayer,to help reduce non-specific signals. Thus, for example, preferredembodiments utilize conductive oligomers that terminate below thesurface of the rest of the monolayer, i.e. below the insulator layer, ifused, or below some fraction of the other conductive oligomers.Similarly, the use of different conductive oligomers may be done tofacilitate monolayer formation, or to make monolayers with alteredproperties.

In a preferred embodiment, the monolayer may further comprise insulatormoieties. By “insulator” herein is meant a substantially nonconductingoligomer, preferably linear. By “substantially nonconducting” herein ismeant that the insulator will not transfer electrons at 100 Hz. The rateof electron transfer through the insulator is preferrably slower thanthe rate through the conductive oligomers described herein.

In a preferred embodiment, the insulators have a conductivity, S, ofabout 10⁻⁷ Ω⁻¹cm⁻¹ or lower, with less than about 10⁻⁸ Ω⁻¹cm⁻¹ beingpreferred. See generally Gardner et al., supra.

Generally, insulators are alkyl or heteroalkyl oligomers or moietieswith sigma bonds, although any particular insulator molecule may containaromatic groups or one or more conjugated bonds. By “heteroalkyl” hereinis meant an alkyl group that has at least one heteroatom, i.e. nitrogen,oxygen, sulfur, phosphorus, silicon or boron included in the chain.Alternatively, the insulator may be quite similar to a conductiveoligomer with the addition of one or more heteroatoms or bonds thatserve to inhibit or slow, preferably substantially, electron transfer.

Suitable insulators are known in the art, and include, but are notlimited to, —(CH₂)n—, —(CRH)_(n)—, and —(CR₂)_(n)—, ethylene glycol orderivatives using other heteroatoms in place of oxygen, i.e. nitrogen orsulfur (sulfur derivatives are not preferred when the electrode isgold).

As for the conductive oligomers, the insulators may be substituted withR groups as defined herein to alter the packing of the moieties orconductive oligomers on an electrode, the hydrophilicity orhydrophobicity of the insulator, and the flexibility, i.e. therotational, torsional or longitudinal flexibility of the insulator. Forexample, branched alkyl groups may be used. Similarly, the insulatorsmay contain terminal groups, as outlined above, particularly toinfluence the surface of the monolayer.

The length of the species making up the monolayer will vary as needed.As outlined above, it appears that binding is more efficient at adistance from the surface. The attachment linker to which the bindingligands are attached (as outlined below, these can be either insulatorsor conductive oligomers) may be basically the same length as themonolayer forming species or longer than them, resulting in the bindingligands being more accessible to the solvent for binding. In someembodiments, the conductive oligomers to which the binding ligands areattached may be shorter than the monolayer.

As will be appreciated by those in the art, the actual combinations andratios of the different species making up the monolayer can vary widely.Generally, three component systems are preferred, with the first speciescomprising a binding ligand. The second species are the conductiveoligomers, and the third species are insulators. In this embodiment, thefirst species can comprise from about 90% to about 1%, with from about20% to about 40% being preferred, and from about 30% to about 40% beingespecially preferred for small targets and from about 10% to about 20%preferred for larger targets. The second species can comprise from about1% to about 90%, with from about 20% to about 90% being preferred, andfrom about 40% to about 60% being especially preferred. The thirdspecies can comprise from about 1% to about 90%, with from about 20% toabout 40% being preferred, and from about 15% to about 30% beingespecially preferred. Preferred ratios of first:second:third species are2:2:1 for small targets, 1:3:1 for larger targets, with total thiolconcentration in the 500 μM to 1 mM range, and 833 μM being preferred.

In a preferred embodiment, two component systems are used, comprisingthe first and second species. In this embodiment, the first species cancomprise from about 90% to about 1%, with from about 1% to about 40%being preferred, and from about 10% to about 40% being especiallypreferred. The second species can comprise from about 1% to about 90%,with from about 10% to about 60% being preferred, and from about 20% toabout 40% being especially preferred.

The covalent attachment of the conductive oligomers and insulators maybe accomplished in a variety of ways, depending on the electrode and thecomposition of the insulators and conductive oligomers used. In apreferred embodiment, the attachment linkers with covalently attachedbinding ligands as depicted herein are covalently attached to anelectrode. Thus, one end or terminus of the attachment linker isattached to the binding ligand, and the other is attached to anelectrode. In some embodiments it may be desirable to have theattachment linker attached at a position other than a terminus, or evento have a branched attachment linker that is attached to an electrode atone terminus and to two or more nucleosides at other termini, althoughthis is not preferred. Similarly, the attachment linker may be attachedat two sites to the electrode, as is generally depicted in Structures11-13. Generally, some type of linker is used, as depicted below as “A”in Structure 10, where “X” is the conductive oligomer, “I” is aninsulator and the hatched surface is the electrode:

In this embodiment, A is a linker or atom. The choice of “A” will dependin part on the characteristics of the electrode. Thus, for example, Amay be a sulfur moiety when a gold electrode is used.

Alternatively, when metal oxide electrodes are used, A may be a silicon(silane) moiety attached to the oxygen of the oxide (see for exampleChen et al., Langmuir 10:3332-3337 (1994); Lenhard et al., J.Electroanal. Chem. 78:195-201 (1977), both of which are expresslyincorporated by reference). When carbon based electrodes are used, A maybe an amino moiety (preferably a primary amine; see for exampleDeinhammer et al., Langmuir 10:1306-1313 (1994)). Thus, preferred Amoieties include, but are not limited to, silane moieties, sulfurmoieties (including alkyl sulfur moieties), and amino moieties. In apreferred embodiment, epoxide type linkages with redox polymers such asare known in the art are not used.

Although depicted herein as a single moiety, the insulators andconductive oligomers may be attached to the electrode with more than one“A” moiety; the “A” moieties may be the same or different. Thus, forexample, when the electrode is a gold electrode, and “A” is a sulfuratom or moiety, multiple sulfur atoms may be used to attach theconductive oligomer to the electrode, such as is generally depictedbelow in Structures 11,12 and 13. As will be appreciated by those in theart, other such structures can be made. In Structures 11, 12 and 13, theA moiety is just a sulfur atom, but substituted sulfur moieties may alsobe used.

It should also be noted that similar to Structure 13, it may be possibleto have a a conductive oligomer terminating in a single carbon atom withthree sulfur moities attached to the electrode. Additionally, althoughnot always depicted herein, the conductive oligomers and insulators mayalso comprise a “Q” terminal group.

In a preferred embodiment, the electrode is a gold electrode, andattachment is via a sulfur linkage as is well known in the art, i.e. theA moiety is a sulfur atom or moiety. Although the exact characteristicsof the gold-sulfur attachment are not known, this linkage is consideredcovalent for the purposes of this invention. A representative structureis depicted in Structure 14, using the Structure 3 conductive oligomer,although as for all the structures depicted herein, any of theconductive oligomers, or combinations of conductive oligomers, may beused. Similarly, any of the conductive oligomers or insulators may alsocomprise terminal groups as described herein. Structure 14 depicts the“A” linker as comprising just a sulfur atom, although additional atomsmay be present (i.e. linkers from the sulfur to the conductive oligomeror substitution groups). In addition, Structure 14 shows the sulfur atomattached to the Y aromatic group, but as will be appreciated by those inthe art, it may be attached to the B—D group (i.e. an acetylene) aswell.

In a preferred embodiment, the electrode is a carbon electrode, i.e. aglassy carbon electrode, and attachment is via a nitrogen of an aminegroup. A representative structure is depicted in Structure 15. Again,additional atoms may be present, i.e. Z type linkers and/or terminalgroups.

In Structure 16, the oxygen atom is from the oxide of the metal oxideelectrode. The Si atom may also contain other atoms, i.e. be a siliconmoiety containing substitution groups. Other attachments for SAMs toother electrodes are known in the art; see for example Napier et al.,Langmuir, 1997, for attachment to indium tin oxide electrodes, and alsothe chemisorption of phosphates to an indium tin oxide electrode (talkby H. Holden Thorpe, CHI conference, May 4-5, 1998).

As outlined herein, the binding ligand can be attached to the electrodein a variety of ways. In a preferred embodiment, the binding ligand canbe directly attached, as is known in the art, for example see Millan etal., Anal. Chem. 65:2317-2323 (1993); Millan et al., Anal. Chem.662943-2948 (1994); and WO 97/01646, all of which are incorporated byreference.

In a preferred embodiment, the binding ligand is attached to theelectrode via an attachment linker, which, similar to a passivationagent, can be either an insulator or a conductive oligomer. Thus, as forthe attachment of ETMs to the binding ligands, the attachment linker andthe binding ligand can comprise functional groups used for attachment,either directly or through an additional linker.

Once made, the compositions find use in a variety of applications. Thepresent invention can be used to monitor ligand-induced folding ofproteins, or ligand-induced conformational changes, for the detection oftarget analytes.

In a preferred embodiment, ligand-induced folding of the binding analytemay be monitored. For example, a preferred embodiment utilizes thehix-induced folding of the DNA-binding domain of hin recombinase (hin)(Sluka et al., Science, 238, 1129-1132, 1987). NMR and circulardichrosim spectroscopies demonstrate that hin remains in a fullyunfolded, random-coil coformation in the absence of its ligand, the 14base pair hix recombinase half-site (Sluka et al., Science,238:1129-1132, 1987; Plaxco, Thesis, California Institute of Technology,1993). Upon addition of DNA containing the hix sequence, thisrandom-coil conformation folds very rapidly (estimated time constant isroughly 100 μs, based on Plaxco, et al., J. Mol. Biol., 277:985-994,1998b) to its native conformation to form a stable protein-DNA complex(Sluka et al., supra). Both solution-phase and solid-state studies canbe performed using the 52 residue hin domain, previously prepared usingautomated chemical synthesis techniques (Feng et al., J. Mol. Biol.,232:82-986, 1993). An X-ray crystallographic structure of the hin-hixcomplex has been reported (Feng et al., Science, 263:348-355, 1994),which will allow for the rational placement of redox active metalcenters (ETMs). An additional advantage of the hin-hix system is theease with which the appropriate ligand can be synthesized. The targetligand, the 14 base pair hix half-site (in the context of a somewhatlarger element of DNA to ensure stability) can be prepared by automatedchemical synthesis with minimal purification (Feng et al., J. Mol.Biol., 232: 982-986, 1993).

A variety of ETM-modified hin sequences can be made for solution andsolid state studies of electron transfer (ET). In a preferredembodiment, two site-specific, spectroscopically unique, ruthenium-baseddonor and acceptor complexes are made by including differentiallyprotected metal-binding histidine residues pairs in the protein. Hinmutants can be labeled at several different sites to result inmodifications that do not significantly affect the folding of theprotein and which provide the largest possible change in electrontransfer rates upon complex formation. The solved crystal structure ofthe complex allows the rational selection of metal binding sites inorder to optimize ET rate changes (Feng et al., Science, 263:348-355,1994). Using this structure, a number of relatively sequence-distantpairs (i.e. residues relatively well separated in the unfolded state)have been selected which are solvent exposed, appear to benon-structural in nature and which are in close proximity in the foldedcomplex (see FIG. 2). The ability to chemically synthesize hin providesa facile route for the site-specific attachment of ETMs at thesepositions; see Mutz et al., Proc. Natl. Acad. Sci. USA, 93:9521-9526,1996; Dahiyat et al., Inorg. Chimica Acta, 243:207-212, 1996. Thewild-type hin sequence contains two histidine residues, which aresuitable for this attachment. These residues are highly solvent exposedand appear to lack any sturucturally critical roles (FIG. 2). Inaddition to this naturally occuring histidine pair, one or both of theseresidues can be mutated, for example to serine (a residues of similarhydrophobicity and conformational preferences) in order to generateother redox labeling site pairs by the addition of new histidines to thesequence (Dahiyat et al., Inorg. Chimica Acta, 243: 207-212, 1996). Ofparticular importance as a potential labeling site are residues near thecarboxy terminus of the domain. Previous research demonstrates thatbulky iron-EDTA groups can be affixed to the carboxy-terminus and thatthe carboxy-terminal six residues of the domain can be deleted withoutreducing ligand affinity (Mack and Dervan, Biochemistry, 31:9399-9400,1992), showing that the addition of a terminal ETM will notsignificantly affect ligand-induced folding. In addition, a number ofdifferentially protected fmoc-histidines are commercially availablewhich greatly facilitiates the specific labeling of the two sites.

In a preferred embodiment, the ETMs comprise a ruthenium electron donor,[Ru(NH3)4(pyridine)(his)]2+ and a ruthenium acceptor,{ru(bpy)2(imidazole)(his)]3+, which will spontaneously form from readilyavailable precursor ruthenium compounds when liganded to specifichistidine residues in the protein (Dahiyat, Inorg. Chimica Acta,243:207-212, 1996; Mutz et al., Proc. Natl. Acad. Sci. USA,93:9521-9526, 1996). Very similar chemistry can be used incorporate ETMscontaining other metals, such as listed above, and secondary ligandsthus providing the ability to “fine-tune” the redox potential andreorganization energies of the labeled protein for the optimization ofthe ET signal. In particular, the use of more “hydrophobic” ETMs, suchas ferrocene, can generate a significant, folding-induced change inreorganization energy if the hydrophobic groups in unfolded hinpartially solvate the metal center. Such solvation would be abolished bythe folding of hin and thus leads to a significant, detectablealteration of ET. Previous reports suggest that the purification ofdifferentially labeled proteins from unreacted or partially-reactedcontaminants can be easily performed using reverse-phase HPLC (e.g.Mutz, et al., supra; Dahiyat, et al., supra). ET may be monitored in avariety of ways, as outlined herein, with a preferred method utilizingflash-photolysis based electron transfer techniques (Mutz et al., supra;Meade and Kayyem, Ang. Chemie, 34:352-354, 1996).

An additional preferred embodiment is directed to detectingligand-induced conformational changes, which are generally not as largeas those of folding (Uversky and Narizhnev, supra). A preferredembodiment utilizes the ligand-induced conformational changes incalmodulin and the RNA-binding domain of HIV trans-activating protein(Tat). These ligand-induced conformational changes are more subtle thanthe ligand-induced folding of hin (Seeger et al., J. Am. Chem. Soc.,119:5118-5125, 1997; Mujeeb et al., J. Biomol. Struct. Dynam.,13:649-660, 1996), and the systems are structurally solved (e.g. Ban etal., Acta Cryst. D., 50:50-63, 1994; Mujeeb et al., supra). Both ofthese lack structurally critical histidines and contain a number ofpotential metal labeling sites straddling the site of the conformationalchange. The ligands (specific decapeptides for calmodulin and the TARRNA sequence for Tat) are easily prepared in large amounts. The Tatligand is expressed at high levels by many retroviruses (Karn et al., J.Karn, Ed., 147-165, IRL Press, 1995) and thus serves as a commerciallyrelevant target analyte.

In addition, as outlined herein, the use of a solid support such as anelectrode enables the use of these binding ligands in an array form. Theuse of oligonucleotide arrays are well known in the art, and similararrays can be made using the binding ligands of the invention. Inaddition, techniques are known for “addressing” locations within anelectrode and for the surface modification of electrodes. Thus, in apreferred embodiment, arrays of different binding ligands are laid downon the electrode, each of which are covalently attached to the electrodevia an attachment linker. In this embodiment, the number of differentspecies of binding ligands may vary widely, from one to thousands, withfrom about 4 to about 100,000 being preferred, and from about 10 toabout 10,000 being particularly preferred.

Once the assay complexes of the invention are made, that minimallycomprise a target analyte and a binding ligand, detection proceeds. In asolution-based system, this can be done in a variety of ways, includingflash photolysis.

In a preferred embodiment, when an electrode is used, detection proceedswith electronic initiation. Without being limited by the mechanism ortheory, detection is based on the transfer of electrons between theETMs; either from a donor ro an acceptor, in the embodiment thatutilizes solution based detection, or from the ETM to the electrode.

Detection of electron transfer, i.e. the presence of the ETMs, isgenerally initiated electronically, with voltage being preferred. Apotential is applied to the assay complex. Precise control andvariations in the applied potential can be via a potentiostat and eithera three electrode system (one reference, one sample (or working) and onecounter electrode) or a two electrode system (one sample and one counterelectrode). This allows matching of applied potential to peak potentialof the system which depends in part on the choice of ETMs and in part onthe conductive oligomer used, the composition and integrity of themonolayer, and what type of reference electrode is used. As describedherein, ferrocene is a preferred ETM.

Electron transfer or the presence of the ETMs at the surface of themonolayer can be detected in a variety of ways. A variety of detectionmethods may be used, including, but not limited to, optical detection(as a result of spectral changes upon changes in redox states), whichincludes fluorescence, phosphorescence, luminiscence, chemiluminescence,electrochemiluminescence, and refractive index; and electronicdetection, including, but not limited to, amperommetry, voltammetry,capacitance and impedence. These methods include time or frequencydependent methods based on AC or DC currents, pulsed methods, lock-intechniques, filtering (high pass, low pass, band pass), andtime-resolved techniques including time-resolved fluorescence.

In one embodiment, the efficient transfer of electrons from the ETM tothe electrode results in stereotyped changes in the redox state of theETM. With many ETMs including the complexes of ruthenium containingbipyridine, pyridine and imidazole rings, these changes in redox stateare associated with changes in spectral properties. Significantdifferences in absorbance are observed between reduced and oxidizedstates for these molecules. See for example Fabbrizzi et al., Chem. Soc.Rev. 1995 pp 197-202). These differences can be monitored using aspectrophotometer or simple photomultiplier tube device.

In this embodiment, possible electron donors and acceptors include allthe derivatives listed above for photoactivation or initiation.Preferred electron donors and acceptors have characteristically largespectral changes upon oxidation and reduction resulting in highlysensitive monitoring of electron transfer. Such examples includeRu(NH₃)₄py and Ru(bpy)₂im as preferred examples. It should be understoodthat only the donor or acceptor that is being monitored by absorbanceneed have ideal spectral characteristics.

In a preferred embodiment, the electron transfer is detectedfluorometrically. Numerous transition metal complexes, including thoseof ruthenium, have distinct fluorescence properties. Therefore, thechange in redox state of the electron donors and electron acceptorsattached to the nucleic acid can be monitored very sensitively usingfluorescence, for example with Ru(4,7-biphenyl₂-phenanthroline)₃ ²⁺. Theproduction of this compound can be easily measured using standardfluorescence assay techniques. For example, laser induced fluorescencecan be recorded in a standard single cell fluorimeter, a flow through“on-line” fluorimeter (such as those attached to a chromatographysystem) or a multi-sample “plate-reader” similar to those marketed for96-well immuno assays.

Alternatively, fluorescence can be measured using fiber optic sensorswith nucleic acid probes in solution or attached to the fiber optic.Fluorescence is monitored using a photomultiplier tube or other lightdetection instrument attached to the fiber optic. The advantage of thissystem is the extremely small volumes of sample that can be assayed.

In addition, scanning fluorescence detectors such as the Fluorlmagersold by Molecular Dynamics are ideally suited to monitoring thefluorescence of modified nucleic acid molecules arrayed on solidsurfaces. The advantage of this system is the large number of electrontransfer probes that can be scanned at once using chips covered withthousands of distinct nucleic acid probes.

Many transition metal complexes display fluorescence with large Stokesshifts. Suitable examples include bis- and trisphenanthroline complexesand bis- and trisbipyridyl complexes of transition metals such asruthenium (see Juris, A., Balzani, V., et. al. Coord. Chem. Rev., V. 84,p. 85-277, 1988). Preferred examples display efficient fluorescence(reasonably high quantum yields) as well as low reorganization energies.These include Ru(4,7-biphenyl₂-phenanthroline)₃ ²⁺,Ru(4,4′-diphenyl-2,2′-bipyridine)₃ ²⁺ and platinum complexes (seeCummings et al., J. Am. Chem. Soc. 118:1949-1960 (1996), incorporated byreference). Alternatively, a reduction in fluorescence associated withhybridization can be measured using these systems.

In a further embodiment, electrochemiluminescence is used as the basisof the electron transfer detection. With some ETMs such as Ru²⁺(bpy)₃,direct luminescence accompanies excited state decay. Changes in thisproperty are associated with nucleic acid hybridization and can bemonitored with a simple photomultiplier tube arrangement (see Blackburn,G. F. Clin. Chem. 37: 1534-1539 (1991); and Juris et al., supra.

In a preferred embodiment, electronic detection is used, includingamperommetry, voltammetry, capacitance, and impedence. Suitabletechniques include, but are not limited to, electrogravimetry;coulometry (including controlled potential coulometry and constantcurrent coulometry); voltametry (cyclic voltametry, pulse voltametry(normal pulse voltametry, square wave voltametry, differential pulsevoltametry, Osteryoung square wave voltametry, and coulostatic pulsetechniques); stripping analysis (aniodic stripping analysis, cathiodicstripping analysis, square wave stripping voltammetry); conductancemeasurements (electrolytic conductance, direct analysis); time-dependentelectrochemical analyses (chronoamperometry, chronopotentiometry, cyclicchronopotentiometry and amperometry, AC polography, chronogalvametry,and chronocoulometry); AC impedance measurement; capacitancemeasurement; AC voltametry; and photoelectrochemistry.

In a preferred embodiment, monitoring electron transfer is viaamperometric detection. This method of detection involves applying apotential (as compared to a separate reference electrode) between thenucleic acid-conjugated electrode and a reference (counter) electrode inthe sample containing target genes of interest. Electron transfer ofdiffering efficiencies is induced in samples in the presence or absenceof target analyte which can result in different currents.

The device for measuring electron transfer amperometrically involvessensitive current detection and includes a means of controlling thevoltage potential, usually a potentiostat. This voltage is optimizedwith reference to the potential of the electron donating complex on thelabel probe. Possible electron donating complexes include thosepreviously mentioned with complexes of iron, osmium, platinum, cobalt,rhenium and ruthenium being preferred and complexes of iron being mostpreferred.

In a preferred embodiment, alternative electron detection modes areutilized. For example, potentiometric (or voltammetric) measurementsinvolve non-faradaic (no net current flow) processes and are utilizedtraditionally in pH and other ion detectors. Similar sensors are used tomonitor electron transfer between the ETM and the electrode. Inaddition, other properties of insulators (such as resistance) and ofconductors (such as conductivity, impedance and capicitance) could beused to monitor electron transfer between ETM and the electrode.Finally, any system that generates a current (such as electron transfer)also generates a small magnetic field, which may be monitored in someembodiments.

It should be understood that one benefit of the fast rates of electrontransfer observed in the compositions of the invention is that timeresolution can greatly enhance the signal-to-noise results of monitorsbased on absorbance, fluorescence and electronic current. The fast ratesof electron transfer of the present invention result both in highsignals and stereotyped delays between electron transfer initiation andcompletion. By amplifying signals of particular delays, such as throughthe use of pulsed initiation of electron transfer and “lock-in”amplifiers of detection, and Fourier transforms.

In a preferred embodiment, electron transfer is initiated usingalternating current (AC) methods. Without being bound by theory, itappears that ETMs, bound to an electrode, generally respond similarly toan AC voltage across a circuit containing resistors and capacitors.Basically, any methods which enable the determination of the nature ofthese complexes, which act as a resistor and capacitor, can be used asthe basis of detection. Surprisingly, traditional electrochemicaltheory, such as exemplified in Laviron et al., J. Electroanal. Chem.97:135 (1979) and Laviron et al., J. Electroanal. Chem. 105:35 (1979),both of which are incorporated by reference, do not accurately model thesystems described herein, except for very small E_(AC) (less than 10 mV)and relatively large numbers of molecules. That is, the AC current (I)is not accurately described by Laviron's equation. This may be due inpart to the fact that this theory assumes an unlimited source and sinkof electrons, which is not true in the present systems.

Accordingly, alternate equations were developed, using the Nernstequation and first principles to develop a model which more closelysimulates the results. This was derived as follows. The Nernst equation,Equation 1 below, describes the ratio of oxidized (O) to reduced (R)molecules (number of molecules=n) at any given voltage and temperature,since not every molecule gets oxidized at the same oxidation potential.$\begin{matrix}{{{Equation}\quad 1}{E_{DC} = {E_{0} + {\frac{RT}{nF}\quad \ln \quad \frac{\lbrack O\rbrack}{\lbrack R\rbrack}}}}} & (1)\end{matrix}$

E_(DC) is the electrode potential, E₀ is the formal potential of themetal complex, R is the gas constant, T is the temperature in degreesKelvin, n is the number of electrons transferred, F is faraday'sconstant, [O] is the concentration of oxidized molecules and [R] is theconcentration of reduced molecules.

The Nernst equation can be rearranged as shown in Equations 2 and 3:$\begin{matrix}{{{Equation}\quad 2}{{E_{DC} - E_{0}} = {\frac{RT}{nF}\quad \ln \quad \frac{\lbrack O\rbrack}{\lbrack R\rbrack}}}} & (2)\end{matrix}$

E_(DC) is the DC component of the potential. $\begin{matrix}{{{Equation}\quad 3}{\exp^{\frac{nF}{RT}{({E_{DC} - E_{0}})}} = \frac{\lbrack O\rbrack}{\lbrack R\rbrack}}} & (3)\end{matrix}$

Equation 3 can be rearranged as follows, using normalization of theconcentration to equal 1 for simplicity, as shown in Equations 4, 5 and6. This requires the subsequent multiplication by the total number ofmolecules.

[O]+[R]=1  Equation 4

[O]=1−[R]  Equation 5

[R]=1−[O]  Equation 6

Plugging Equation 5 and 6 into Equation 3, and the fact that nF/RTequals 38.9 V⁻¹, for n=1 , gives Equations 7 and 8, which define [O] and[R], respectively: $\begin{matrix}{{{Equation}\quad {7\lbrack O\rbrack}} = \frac{\exp^{38.9\quad {({E - E_{0}})}}}{1 + \exp^{38.9\quad {({E - E_{0}})}}}} & (4) \\{{{Equation}\quad {8\lbrack R\rbrack}} = \frac{1}{1 + \exp^{38.9\quad {({E - E_{0}})}}}} & (5)\end{matrix}$

Taking into consideration the generation of an AC faradaic current, theratio of [O]/[R] at any given potential must be evaluated. At aparticular E_(DC) with an applied E_(AC), as is generally describedherein, at the apex of the E_(AC) more molecules will be in the oxidizedstate, since the voltage on the surface is now (E_(DC)+E_(AC)); at thebottom, more will be reduced since the voltage is lower. Therefore, theAC current at a given E_(DC) will be dictated by both the AC and DCvoltages, as well as the shape of the Nernstian curve. Specifically, ifthe number of oxidized molecules at the bottom of the AC cycle issubtracted from the amount at the top of the AC cycle, the total changein a given AC cycle is obtained, as is generally described by Equation9. Dividing by 2 then gives the AC amplitude. $\begin{matrix}{i_{AC} \cong \frac{\left( {{electrons}\quad {{at}\quad\left\lbrack {E_{DC} + E_{AC}} \right\rbrack}} \right) - \left( {{electrons}\quad {{at}\quad\left\lbrack {E_{DC} - E_{AC}} \right\rbrack}} \right)}{2}} & {{Equation}\quad 9}\end{matrix}$

Equation 10 thus describes the AC current which should result:

i _(AC) =C ₀ Fω½([O]_(E) _(DC) _(+E) _(AC) −[O] _(E) _(DC) _(−E) _(AC))(6)  Equation 10

As depicted in Equation 11, the total AC current will be the number ofredox molecules C), times faraday's constant (F), times the AC frequency(ω), times 0.5 (to take into account the AC amplitude), times the ratiosderived above in Equation 7. The AC voltage is approximated by theaverage, E_(AC)2/π. $\begin{matrix}{{{Equation}\quad 11}{i_{AC} = {{\frac{C_{0}F\quad \omega}{2}\quad \left( \frac{\exp^{38.9\quad\lbrack{E_{DC} + \frac{2E_{AC}}{\pi} - E_{0}}\rbrack}}{1 + \exp^{38.9\quad\lbrack{E_{DC} + \frac{2E_{AC}}{\pi} - E_{0}}\rbrack}} \right)} - \frac{\exp^{38.9\quad\lbrack{E_{DC} - \frac{2E_{AC}}{\pi} - E_{0}}\rbrack}}{1 + \exp^{38.9\quad\lbrack{E_{DC} - \frac{2E_{AC}}{\pi} - E_{0}}\rbrack}}}}} & (7)\end{matrix}$

Using Equation 11, simulations were generated using increasingoverpotential (AC voltage). FIG. 22A of PCT US97/20014 shows one ofthese simulations, while FIG. 22B depicts a simulation based ontraditional theory. FIGS. 23A and 23B depicts actual experimental datausing the Fc-wire of Example 7 of PCT US97/20014 plotted with thesimulation, and shows that the model fits the experimental data verywell. In some cases the current is smaller than predicted, however thishas been shown to be caused by ferrocene degradation which may beremedied in a number of ways. However, Equation 11 does not incorporatethe effect of electron transfer rate nor of instrument factors. Electrontransfer rate is important when the rate is close to or lower than theapplied frequency. Thus, the true i_(AC) should be a function of allthree, as depicted in Equation 12.

i _(AC) =f(Nernst factors)f(k _(ET))f(instrument factors)  Equation 12

These equations can be used to model and predict the expected ACcurrents in systems which use input signals comprising both AC and DCcomponents. As outlined above, traditional theory surprisingly does notmodel these systems at all, except for very low voltages.

In general, non-specifically bound analytes show differences inimpedance (i.e. higher impedances) than when the assay complexescontaining the ETMs are specifically bound in the correct orientation.In a preferred embodiment, the non-specifically bound material is washedaway, resulting in an effective impedance of infinity. Thus, ACdetection gives several advantages as is generally discussed below,including an increase in sensitivity, and the ability to “filter out”background noise. In particular, changes in impedance (including, forexample, bulk impedance) as between non-specific binding ofETM-containing probes and target-specific assay complex formation may bemonitored.

Accordingly, when using AC initiation and detection methods, thefrequency response of the system changes as a result of the presence ofthe ETM. By “frequency response” herein is meant a modification ofsignals as a result of electron transfer between the electrode and theETM. This modification is different depending on signal frequency. Afrequency response includes AC currents at one or more frequencies,phase shifts, DC offset voltages, faradaic impedance, etc.

Once the assay complex including the target analyte and binding ligandis made, a first input electrical signal is then applied to the system,preferably via at least the sample electrode (containing the complexesof the invention) and the counter electrode, to initiate electrontransfer between the electrode and the ETM. Three electrode systems mayalso be used, with the voltage applied to the reference and workingelectrodes. The first input signal comprises at least an AC component.The AC component may be of variable amplitude and frequency. Generally,for use in the present methods, the AC amplitude ranges from about 1 mVto about 1.1 V, with from about 10 mV to about 800 mV being preferred,and from about 10 mV to about 500 mV being especially preferred. The ACfrequency ranges from about 0.01 Hz to about 100 MHz, with from about 10Hz to about 10 MHz being preferred, and from about 100 Hz to about 20MHz being especially preferred.

The use of combinations of AC and DC signals gives a variety ofadvantages, including surprising sensitivity and signal maximization.

In a preferred embodiment, the first input signal comprises a DCcomponent and an AC component. That is, a DC offset voltage between thesample and counter electrodes is swept through the electrochemicalpotential of the ETM (for example, when ferrocene is used, the sweep isgenerally from 0 to 500 mV) (or alternatively, the working electrode isgrounded and the reference electrode is swept from 0 to −500 mV). Thesweep is used to identify the DC voltage at which the maximum responseof the system is seen. This is generally at or about the electrochemicalpotential of the ETM. Once this voltage is determined, either a sweep orone or more uniform DC offset voltages may be used. DC offset voltagesof from about −1 V to about +1.1 V are preferred, with from about −500mV to about +800 mV being especially preferred, and from about −300 mVto about 500 mV being particularly preferred. In a preferred embodiment,the DC offset voltage is not zero. On top of the DC offset voltage, anAC signal component of variable amplitude and frequency is applied. Ifthe ETM is present, and can respond to the AC perturbation, an ACcurrent will be produced due to electron transfer between the electrodeand the ETM.

For defined systems, it may be sufficient to apply a single input signalto differentiate between the presence and absence of the ETM.Alternatively, a plurality of input signals are applied. As outlinedherein, this may take a variety of forms, including using multiplefrequencies, multiple DC offset voltages, or multiple AC amplitudes, orcombinations of any or all of these.

Thus, in a preferred embodiment, multiple DC offset voltages are used,although as outlined above, DC voltage sweeps are preferred. This may bedone at a single frequency, or at two or more frequencies.

In a preferred embodiment, the AC amplitude is varied. Without beingbound by theory, it appears that increasing the amplitude increases thedriving force. Thus, higher amplitudes, which result in higheroverpotentials give faster rates of electron transfer. Thus, generally,the same system gives an improved response (i.e. higher output signals)at any single frequency through the use of higher overpotentials at thatfrequency. Thus, the amplitude may be increased at high frequencies toincrease the rate of electron transfer through the system, resulting ingreater sensitivity. In addition, this may be used, for example, toinduce responses in slower systems such as those that do not possessoptimal spacing configurations.

In a preferred embodiment, measurements of the system are taken at atleast two separate amplitudes or overpotentials, with measurements at aplurality of amplitudes being preferred. As noted above, changes inresponse as a result of changes in amplitude may form the basis ofidentification, calibration and quantification of the system. Inaddition, one or more AC frequencies can be used as well.

In a preferred embodiment, the AC frequency is varied. At differentfrequencies, different molecules respond in different ways. As will beappreciated by those in the art, increasing the frequency generallyincreases the output current. However, when the frequency is greaterthan the rate at which electrons may travel between the electrode andthe ETM, higher frequencies result in a loss or decrease of outputsignal. At some point, the frequency will be greater than the rate ofelectron transfer between the ETM and the electrode, and then the outputsignal will also drop.

In one embodiment, detection utilizes a single measurement of outputsignal at a single frequency. That is, the frequency response of thesystem in the absence of target sequence, and thus the absence of labelprobe containing ETMs, can be previously determined to be very low at aparticular high frequency. Using this information, any response at aparticular frequency, will show the presence of the assay complex. Thatis, any response at a particular frequency is characteristic of theassay complex. Thus, it may only be necessary to use a single input highfrequency, and any changes in frequency response is an indication thatthe ETM is present, and thus that the target sequence is present.

In addition, the use of AC techniques allows the significant reductionof background signals at any single frequency due to entities other thanthe ETMs, i.e. “locking out” or “filtering” unwanted signals. That is,the frequency response of a charge carrier or redox active molecule insolution will be limited by its diffusion coefficient and chargetransfer coefficient. Accordingly, at high frequencies, a charge carriermay not diffuse rapidly enough to transfer its charge to the electrode,and/or the charge transfer kinetics may not be fast enough. This isparticularly significant in embodiments that do not have goodmonolayers, i.e. have partial or insufficient monolayers, i.e. where thesolvent is accessible to the electrode. As outlined above, in DCtechniques, the presence of “holes” where the electrode is accessible tothe solvent can result in solvent charge carriers “short circuiting” thesystem, i.e. the reach the electrode and generate background signal.However, using the present AC techniques, one or more frequencies can bechosen that prevent a frequency response of one or more charge carriersin solution, whether or not a monolayer is present. This is particularlysignificant since many biological fluids such as blood containsignificant amounts of redox active molecules which can interfere withamperometric detection methods.

In a preferred embodiment, measurements of the system are taken at atleast two separate frequencies, with measurements at a plurality offrequencies being preferred. A plurality of frequencies includes a scan.For example, measuring the output signal, e.g., the AC current, at a lowinput frequency such as 1-20 Hz, and comparing the response to theoutput signal at high frequency such as 10-100 kHz will show a frequencyresponse difference between the presence and absence of the ETM. In apreferred embodiment, the frequency response is determined at at leasttwo, preferably at least about five, and more preferably at least aboutten frequencies.

After transmitting the input signal to initiate electron transfer, anoutput signal is received or detected. The presence and magnitude of theoutput signal will depend on a number of factors, including theoverpotential/amplitude of the input signal; the frequency of the inputAC signal; the composition of the intervening medium; the DC offset; theenvironment of the system; the nature of the ETM; the solvent; and thetype and concentration of salt. At a given input signal, the presenceand magnitude of the output signal will depend in general on thepresence or absence of the ETM, the placement and distance of the ETMfrom the surface of the monolayer and the character of the input signal.In some embodiments, it may be possible to distinguish betweennon-specific binding probes and the formation of target specific assaycomplexes on the basis of impedance.

In a preferred embodiment, the output signal comprises an AC current. Asoutlined above, the magnitude of the output current will depend on anumber of parameters. By varying these parameters, the system may beoptimized in a number of ways.

In general, AC currents generated in the present invention range fromabout 1 femptoamp to about 1 milliamp, with currents from about 50femptoamps to about 100 microamps being preferred, and from about 1picoamp to about 1 microamp being especially preferred.

In a preferred embodiment, the output signal is phase shifted in the ACcomponent relative to the input signal. Without being bound by theory,it appears that the systems of the present invention may be sufficientlyuniform to allow phase-shifting based detection. That is, the complexbiomolecules of the invention through which electron transfer occursreact to the AC input in a homogeneous manner, similar to standardelectronic components, such that a phase shift can be determined. Thismay serve as the basis of detection between the presence and absence ofthe ETM, and/or differences between the presence of target-specificassay complexes and non-specific binding to the system components.

The output signal is characteristic of the presence of the ETM; that is,the output signal is characteristic of the presence of thetarget-specific assay complex comprising ETMs. In a preferredembodiment, the basis of the detection is a difference in the faradaicimpedance of the system as a result of the formation of the assaycomplex. Faradaic impedance is the impedance of the system between theelectrode and the ETM. Faradaic impedance is quite different from thebulk or dielectric impedance, which is the impedance of the bulksolution between the electrodes. Many factors may change the faradaicimpedance which may not effect the bulk impedance, and vice versa. Thus,the assay complexes comprising the nucleic acids in this system have acertain faradaic impedance, that will depend on the distance between theETM and the electrode, their electronic properties, and the compositionof the intervening medium, among other things. Of importance in themethods of the invention is that the faradaic impedance between the ETMand the electrode is signficantly different depending on whether theETMs are specifically or non-specifically bound to the electrode.

Accordingly, the present invention further provides apparatus for thedetection of target analytes using AC detection methods. The apparatusincludes a test chamber which has at least a first measuring or sampleelectrode, and a second measuring or counter electrode. Three electrodesystems are also useful. The first and second measuring electrodes arein contact with a test sample receiving region, such that in thepresence of a liquid test sample, the two electrodes may be inelectrical contact.

The apparatus further comprises an AC voltage source electricallyconnected to the test chamber; that is, to the measuring electrodes.Preferably, the AC voltage source is capable of delivering DC offsetvoltage as well.

In a preferred embodiment, the apparatus further comprises a processorcapable of comparing the input signal and the output signal. Theprocessor is coupled to the electrodes and configured to receive anoutput signal, and thus detect the presence of the target analyte.

Thus, the compositions of the present invention may be used in a varietyof research, clinical, quality control, or field testing settings.

All references cited herein are incorporated by reference in theirentirety.

We claim:
 1. A method of detecting the presence of a target analyte comprising: a) binding a target analyte to a binding ligand comprising at least a first electron donor moiety and a second electron acceptor moiety; and b) detecting electron transfer between said donor and said acceptor, wherein there is a change in the amount of electron transfer between said donor and acceptor as a result of altering the structured state of said donor and acceptor, said altering induced by a conformational change in said binding ligand upon binding of said target analyte.
 2. A method according to claim 1 wherein said binding ligand is a protein.
 3. A method according to claim 1 wherein said change is an increase.
 4. A method according to claim 1 wherein said change is a decrease. 